Background: The aim of this study is to investigate the effect of trimethylamine (TMA) and trimethylamine-n-oxide (TMAO) on the contractility of human umbilical artery and the possible mechanisms involved. Methods: Vasoactive responses to TMA and TMAO on human umbilical artery rings were measured in isolated organ baths. Cumulative dose–response curves for TMA and TMAO were obtained before and after incubation with atropine, yohimbine, prazosin, indomethacin, verapamil, and Ca⁺²-free Krebs–Henselite solution. Results: Administration of cumulative TMA and TMAO resulted in dose-dependent contraction at concentrations ranging from 10 to 100 mM on human umbilical artery rings. TMA-induced contractions were more potent than TMAO-induced contractions (TMA: −logEC50 = 1.00 ± 0.02, TMAO: −logEC50 = 0.57 ± 0.02). Contraction responses to TMA were significantly lower in the presence of verapamil and in the absence of external Ca⁺² (p < 0.001, p < 0.05, respectively). Conclusion: Our results showed that TMA and TMAO caused vasoconstriction in isolated human umbilical artery rings. Our findings also indicated that TMA but not TMAO-induced vasoconstriction was partially dependent on extracellular Ca²⁺ and calcium influx through L-type Ca²⁺ channels. Our results suggest that TMA and TMAO may have the potential to contribute to cardiovascular diseases through their direct effect on vascular contractility in human arteries.